In 1976, Martin et al. (J. Pharmacol. Exp. Ther. 1976, 197, 517–32) postulated that sigma receptors account for the actions of (+/−)-SKF 10,047 (N-allyl-normetazocine) and related racemic benzomorphans. These compounds produce a spectrum of behaviors in the dog referred to as canine delirium and have psychotomimetic effects in humans. Great interest in the hypothesis of Martin et al. concerning sigma receptors led to intense scrutiny of (+/−)-SKF 10,047. Ten years of additional research revealed that (+/−)-SKF 10,047 binds to three types of receptors: (−)-SKF 10,047 binds primarily to mu and kappa opiate receptors; (+)-SKF 10,047 binds to PCP receptors and to a unique site that retains the designation sigma receptor (See Quirion et al. Trends Neurosci. 1987, 10, 444–46). Sigma receptors have also been called haloperidol-sensitive sigma receptors, etorphine-inaccessible sigma receptors, and naloxone-inaccessible sigma receptors (See Walker et al. Neurology 1988, 38, 961–65).
Sigma receptors were originally thought to be a type of opiate receptor, but two subsequent findings convincingly demonstrated that this characterization was incorrect: (a) whereas opiate receptors are enantioselective for the (−)-isomers of opium-derived narcotics, narcotic antagonists, and their congeners, sigma receptors are enantioselective for the (+)-isomers; and (b) naloxone is ineffective against both the in vivo and in vitro effects of sigma ligands. Therefore, it became clear that the sigma receptor is not a type of opiate receptor.
Initially, investigators asserted that sigma receptors were identical with PCP receptors, based on the displacement of [3H]PCP binding by the prototypic sigma ligand (+/−)-SKF 10,047. For this reason, sigma receptors were sometimes called “sigma opiate/PCP receptors”. However, the drug selectivity pattern of [3H](+)-SKF 10,047 differs from that of [3H]PCP, showing that these substances bind to different receptors. For example, antipsychotic drugs (such as haloperidol) potently displace [3H](+)-SKF 10,047 binding, but they are weak or inactive against [3H]PCP binding. Conversely, PCP is weak antagonist of [3H]haloperidol binding.
Sigma and PCP receptors may also be differentiated by their distinct anatomical distributions, because [3H](+)-SKF 10,047 and [3H]PCP-binding sites are concentrated in different brain areas. Tam pointed out additional differences between [3H]PCP binding and [3H](+)-SKF 10,047 binding: the sensitivity of [3H]PCP binding to sodium ions; and the low affinity and small stereoselectivity shown by PCP receptors toward (+)-SKF 10,047 and (+)-ethylketocyclazocine. These findings showed that [3H](+)-SKF 10,047 binds to two distinct sites: a haloperidol-sensitive site (subsequently called the sigma receptor) and a PCP-sensitive site, subsequently called the PCP receptor.
Radioligand-binding studies revealed that many antipsychotic drugs bind to sigma receptors with high affinity. Haloperidol is among the most potent inhibitors of [3H](+)-SKF 10,047 binding, having a Ki of 4 nM (Itzhak, Y. Life Sci. 1988, 42, 745–52). Other antipsychotic drugs that possess moderate (Ki<1000 nM) to high potency include perphenazine, (−)-butaclamol, acetophenazine, trifluoperazine, molindone, pimozide, thioridazine, and chlorpromazine (Tam and Cook Proc. Natl. Acad. Sci. USA 1984, 81, 5618–21). The connection between sigma receptors and antipsychotic drugs was further strengthened by the finding that [3H]haloperidol binding is strongly reduced by the sigma ligands (+)-SKF 10,047 (+)-pentazocine, and (+)-cyclazocine. In fact, the sigma ligand (+)-pentazocine displaces [3H]haloperidol from its binding sites in guinea pig brain about 10 times more potently than the dopamine ligand spiperone.
Furthermore, sigma receptors and kappa receptors have been shown to bind the same opiates. However, kappa receptors prefer one stereoisomer, and sigma receptors prefer the other. Whereas kappa opiate receptors bind (−)-benzomorphans, sigma receptors bind (+)-benzomorphans. Examples are (−)-SKF 10,047, (−)-pentazocine, and (−)-SKF 10,047, (−)-pentazocine, (−)-cyclazocine, and (−)-ethylketocyclazoine, which bind to kappa opiate receptors; their (+)-enantiomers bind to sigma receptors. Another example of this distinction is found with cis and trans isomers of U50,488. Whereas the trans isomers show preference for kappa opiate receptors, the cis isomers show preference for sigma receptors. Thus, in two chemically unrelated classes of compounds, different isomers show preference for kappa or sigma receptors. These results suggest a complimentarily between the topography of the binding sites of the kappa opiate and the sigma receptor.
Haloperidol exhibits its highest affinity to the sigma site, which is distinct from the classical opiate or phencyclidine sites. See Bartoszyk, G. D. et al. CNS Drug Reviews 1996, 2, 175–94. Functional connections between sigma receptors and dopaminergic neurons in mesolimbic and cortical areas have been identified, and the involvement of sigma sites in the action of antipsychotic drugs has been shown in animal experiments. Further evidence for the significance of sigma sites in schizophrenia comes from investigations showing that benzomorphans cause symptoms that resemble schizophrenia in humans. Finally, several post-mortem studies have shown that the number of sigma binding sites in cortical and cerebellar regions is reduced in schizophrenic patients. Some attempts have been made to develop novel antipsychotic drugs with selective affinity for the sigma receptor that would not have the extrapyramidal side effects (EPS) common to the classic neuroleptics. Among these drugs, rimcazole initially has been shown to have some efficacy in humans. However, such drugs retain EPS potential, and selective sigma ligands have ultimately not shown convincing antipsychotic efficacy in clinical trials. Drugs with greater selectivity for sigma receptors, or subtypes thereof, or drugs with higher intrinsic activity hold promise, e.g., as antipsychotics.
Following the discovery that sigma receptors bind antipsychotic drugs came the expected interest in the possible clinical significance of sigma ligands. Here the question of which effects of antipsychotic drugs may be mediated by sigma receptors becomes the central focus. The high concentration of sigma receptors in the motor system immediately raised the issue of the motor side effects of antipsychotic drugs. Simultaneously, the antipsychotic activity of sigma-active drugs such as haloperidol, coupled with the sigma-activity of rimcazole (a putative antipsychotic), raised the important question of the possibility of novel sigma-binding antipsychotic drugs.
Several attempts have been made to formulate models of the sigma receptor that can explain the SAR data for various classes of sigma ligands. Largent et al. (Mol. Pharmacol. 1987, 32, 772–84) performed conformational calculations on a total of 10 compounds, which included phenothiazines and other structures, in an attempt to determine the interatomic distances between the N-(aromatic plane) and N-(polar function). The calculated energy-minimized conformations of (−)-cyclazocine, cis- and trans-clopenthixol, haloperidol, and (+)-dexclamol were found to match their X-ray crystal structure conformations. This study indicated several structural requirements for sigma binding. First, the primary pharmacophore at sigma sites appears to be the 3- or 4-phenylpiperidine moiety, which is present in most compounds showing high affinity for sigma receptors. Second, affinity is greatly influenced by large hydrophobic N-alkyl substituents. Third, compounds from many different structural classes exhibit substantial affinity for sigma receptors, indicating that certain interatomic distances are not subject to rigid constraint (e.g., N to aromatic ring).
In a markedly different approach to modeling the sigma receptor, Bowen et al. (Eur. J. Pharmacol. 1989, 163, 309–18) found evidence supporting a model of distinct, allosterically-coupled binding domains for non-benzomorphan sigma ligands and sigma-related (+)-benzomorphans. Studies of the sensitivity of rat brain sigma receptors to UV irradiation revealed unusual binding interactions of the various radiolabeled sigma probes. This study suggested that benzomorphan and non-benzomorphan sigma ligands interact with different sites on the receptor macromolecule that can be distinguished by differences in their sensitivities to UV light.
A model consistent with all the results cited above, and others not explicitly discussed, is one in which benzomorphans bind to a domain on the receptor macromolecule that is resistant to the effects of UV light. Furthermore, this domain is allosterically coupled to a binding domain for non-benzomorphans. The non-benzomorphan domain is sensitive to UV irradiation, perhaps because of the presence of a UV-sensitive residue such as tryptophan.
At the present time, there does not appear to be a unifying hypothesis capable of reconciling the different topographic and structural models of the sigma receptor. However, the evidence clearly points to interaction of sigma ligands with a heterogeneous population of sites. It is also important to note that one model does not necessarily preclude another. For example, it is conceivable that there are at least two sigma receptor macromolecules, one of which consists of distinct allosterically coupled binding sites, while the other consists of a single ligand binding domain. Additionally, superimposed on these general schemes might be subtle species or tissue differences in the structure of receptor proteins that might affect the ligand-binding profiles.
Sigma receptors are concentrated in (a) brainstem areas that primarily subserve motor functions, (b) certain limbic structures, (c) some predominantly sensory areas, and (d) brain areas associated with endocrine function. See McLean and Weber Neuroscience 1988, 25, 259–69. Sigma receptors are more concentrated in motor areas than in limbic areas. The distribution in the motor system is marked by the high densities found in brainstem motor circuits. For example, the cerebellum and its closely associated circuits, the red nucleus, inferior olive and locus coeruleus, are all rich in sigma receptors. Furthermore, sigma binding is found in cranial nerve nuclei that are rich in motor neurons (facial, motor trigeminal, hypoglossal, and oculomotor, as well as in the anterior horn of the spinal cord. These data form one of several lines of evidence for a function of the sigma receptor in motor function.
Several limbic structures are labeled by sigma radioligands. These areas include the cingulate cortex, lateral and medial septum, hippocampus, hypothalamus, parts of the limbic thalamus, habenula, and anterodorsal nucleus. The presence of sigma receptors in limbic systems might suggest a rule of sigma receptors in emotion and memory. Sigma receptors are found in certain areas that are clearly related to sensory processing. Most notable among these is the heavy labeling of dorsal root ganglia by [3H](+)-3-PPP. See Gundlach et al. J. Neurosci. 1986, 6, 1757–70. The dorsal lateral geniculate and anterior pretectal areas (associated with visual information processing) are also heavily labeled by [3H]DTG.
Although the brain distribution of sigma receptors is unique, some associations with the distribution of cholinergic neurons are notable. See Sofroniew et al. In The Rat Nervous System, vol. 1, pp. 471–85, Academic Press, NY, 1985. For example, sigma receptors are rich in cranial nerve motor nuclei, spinal ventral horns, dorsal diagonal band of Broca, and septal region, all of which possess cholinergic neurons. These two receptor systems do not overlap completely, however, because the caudate, which is rich in acetylcholine, has low levels of sigma receptors.
Sigma receptors are found in many areas of the brain associated with endorcrine function. The heavy labeling over the supraoptic and paraventricular nuclei within the hypothalamus suggests that sigma receptors participate in the regulation of vasopressin (and/or dynorphin) secretion. Dense labeling was also found in the adenohypophysis, suggesting regulation of anterior pituitary hormones. Using [3H](+)-3-PPP, Jansen et al. (Brain Res. 1990, 507, 158–60) demonstrated high levels of sigma receptors in the rat pineal gland, again linking sigma receptors to endocrine function. The relation of sigma receptors to endorcrine function is further supported by the presence of sigma receptors in many peripheral endorcrine tissues.
Cell surface proteins permit intracellular transduction of extracellular signals. Cell surface proteins provide eukaryotic, as well as prokaryotic, cells a means to detect extracellular signals and transduce such signals intracellularly in a manner that ultimately results in a cellular response or a concerted tissue or organ response. Cell surface proteins, by intracellularly transmitting information regarding the extracellular environment via specific intracellular pathways induce an appropriate response to a particular stimulus. The response may be immediate and transient, slow and sustained, or some mixture thereof. By virtue of an array of varied membrane surface proteins, eukaryotic cells are exquisitely sensitive to their environment.
Extracellular signal molecules, such as ligands for the sigma receptor, vasodilators and neurotransmitters, exert their effects, at least in part, via interaction with cell surface proteins. For example, some extracellular signal molecules cause changes in transcription of target gene via changes in the levels of secondary messengers, such as cAMP. Other signals, indirectly alter gene expression by activating the expression of genes, such as immediate-early genes that encode regulatory proteins, which in turn activate expression of other genes that encode transcriptional regulatory proteins. For example, neuron gene expression is modulated by numerous extracellular signals, including neurotransmitters and membrane electrical activity. Transsynaptic signals cause rapid responses in neurons that occur over a period of time ranging from milliseconds, such as the opening of ligand-gated channels, to seconds and minutes, such as second messenger-mediated events. Genes in neural cells that are responsive to transsynaptic stimulation and membrane electrical activity, include genes, called immediate early genes, whose transcription is activated rapidly, within minutes, and transiently (See, e.g. Sheng et al. (1990) Neuron 4: 477–485), and genes whose expression requires protein synthesis and whose expression is induced or altered over the course of hours.
Cell surface-localized receptors are membrane spanning proteins that bind extracellular signalling molecules or changes in the extracellular environment and transmit the signal via signal transduction pathways to effect a cellular response. Cell surface receptors bind circulating signal polypeptides, such as growth factors and hormones, as the initiating step in the induction of numerous intracellular pathways. Receptors are classified on the basis of the particular type of pathway that is induced. Included among these classes of receptors are those that bind growth factors and have intrinsic tyrosine kinase activity, such as the heparin binding growth factor (HBGF) receptors, and those that couple to effector proteins through guanine nucleotide binding regulatory proteins, which are referred to as G protein coupled receptors and G proteins, respectively.
G proteins play a central role in several types of signaling mechanisms (See Gilman, A. G. Annu. Rev. Biochem. 1987, 56, 615–49). In some systems, the first step in the cascade of biochemical events, from the formation of a transmitter-receptor complex to membrane conductance changes, is the coupling of the receptor to a G protein. G proteins play a role in cyclic adenosine monophosphate-related systems, PPI turnover, direct coupling to some ion channels, arachidonic acid-derived systems, and protein translocation (See Casey and Gilman, J. Biol. Chem. 1988, 263, 2577–80).
The G protein transmembrane signaling pathways consist of three proteins: receptors, G proteins and effectors. G proteins, which are the intermediaries in transmembrane signaling pathways, are heterodimers and consist of alpha, beta and gamma subunits. Among the members of a family of G proteins the alpha subunits differ. Functions of G proteins are regulated by the cyclic association of GTP with the alpha subunit followed by hydrolysis of GTP to GDP and dissociation of GDP.
G protein coupled receptors are a diverse class of receptors that mediate signal transduction by binding to G proteins. Signal transduction is initiated via ligand binding to the cell membrane receptor, which stimulates binding of the receptor to the G protein. The receptor G protein interaction releases GDP, which is specifically bound to the G protein, and permits the binding of GTP, which activates the G protein. Activated G protein dissociates from the receptor and activates the effector protein, which regulates the intracellular levels of specific second messengers. Examples of such effector proteins include adenyl cyclase, guanyl cyclase, phospholipase C, and others.
G protein-coupled receptors are known to be inducible. This inducibility was originally described in lower eukaryotes. For example, the cAMP receptor of the cellular slime mold, Dictyostelium, is induced during differentiation (Klein et al., Science 241: 1467–1472 (1988). During the Dictyostelium discoideum differentiation pathway, cAMP, induces high level expression of its G protein-coupled receptor. This receptor transduces the signal to induce the expression of the other genes involved in chemotaxis, which permits multicellular aggregates to align, organize and form stalks (see, Firtel, R. A., et al. Cell 58: 235–239 (1989) and Devreotes, P., Science 245: 1054–1058 (1989)).
Itzhak (See Mol. Pharmacol. 1989, 36, 512–17) reported evidence that sigma receptors interact with G proteins. As observed with other G protein-coupled receptors, guanosine triphosphate and Gpp(NY)p inhibited the binding of [3H](+)-3-PPP to rat brain membranes. Binding of [3H](+)-SKF 19m946 was also inhibited but was less affected than [3H](+)3-PPP binding. Guanosine monophosphate and adenosine triphosphate had no effect on [3H](+)-3-PPP binding, demonstrating specificity for G protein-active guanine nucleotides. Other agents known to affect receptor-G protein coupling also inhibited [3H](+)-3-PPP binding. Treatment of rat brain membranes with either N-ethylmaleimide (a nonselective agent) or pertussis toxin (which selectively alters G proteins) significantly decreased [3H](+)-3-PPP binding. These reagents also eliminated the effect of Gpp(NH)p on [3H](+)-3-PPP binding. These results are similar to those obtained with other G protein-coupled receptors where these reagents are believed to cause uncoupling of the receptor from the G-protein unit.
Taken together, these results strongly suggest that the sigma receptor labeled by [3H](+)-3-PPP can exist in a high and low affinity state, with the high affinity state coupled to a G protein. This suggestion has important. implications for the function of sigma sites, because it suggests that sigma receptors are involved in signal transduction.